The present invention relates to xe2x80x9cgene silencingxe2x80x9d (xe2x80x9cgsxe2x80x9d) in transgenic plants. It employs xe2x80x9camplicon constructsxe2x80x9d, providing in various aspects nucleic acid molecules and vectors, cells and plants containing these, and methods and uses.
xe2x80x9cGene silencingxe2x80x9d is a term generally used to refer to suppression of expression of a gene. The degree of reduction may be so as to totally abolish production of the encoded gene product, but more usually the abolition of expression is partial, with some degree of expression remaining. The term should not therefore be taken to require complete xe2x80x9csilencingxe2x80x9d of expression. It is used herein where convenient because those skilled in the art well understand this.
Transgenes may be used to suppress endogenous plant genes. This was discovered originally when chalcone synthase transgenes in petunia caused suppression of the endogenous chalcone synthase genes (29,35). Subsequently it has been described how many, if not all plant genes can be xe2x80x9csilencedxe2x80x9d by transgenes (11,12,16,17,19,23). Gene silencing requires sequence similarity between the transgene and the gene that becomes silenced (Matzke, M. A. and Matzke, A. J. M. (1995), Trends in Genetics, 11: 1-3). This sequence homology may involve promoter regions or coding regions of the silenced gene (Matzke, M. A. and Matzke, A. J. M. (1993) Annu. Rev. Plant Physiol. Plant Mol. Biol., 44: 53-76, Vaucheret, H. (1993) C. R. Acad. Sci. Paris, 316: 1471-1483, Vaucheret, H. (1994), C. R. Acad. Sci. Paris, 317: 310-323, Baulcombe, D. C. and English, J. J. (1996), Current Opinion In Biotechnology, 7: 173-180, Park, Y-D., et al (1996), Plant J., 9: 183-194). When coding regions are involved the transgene able to cause gene silencing may have been constructed with a promoter that would transcribe either the sense or the antisense orientation of the coding sequence RNA. In at least one example the coding sequence transgene was constructed without a promoter (Van Blokland, R., et al (1994), Plant J., 6: 861-877). It is likely that the various examples of gene silencing involve different mechanisms that are not well understood. In different examples there may be transcriptional or post transcriptional gs (3,4,10,24).
It has also become clear that gene silencing can account for some characteristics of transgenic plants that are not easily reconciled with conventional understanding of genetics. For example the wide variation in transgene expression between sibling lines with a transgene construct is due in part to gene silencing: low expressers are those with a high level of gene silencing whereas high expressers are those in which gene silencing is absent or induced late in plant development (10,13,14). Similarly gene silencing can often explain virus resistance in transgenic lines in which a viral cDNA transgene is expressed at a low level: the gene silencing mechanism acting on RNA inhibits accumulation of both the transgene RNA and the viral RNA (5,10,22).
In principle there is an enormous practical potential of gs for crop improvement. It is possible to silence genes conferring unwanted traits in the plant by transformation with transgene constructs containing elements of these genes. Examples of this type of application include gs of ripening specific genes in tomato to improve processing and handling characteristics of the harvested fruit; gs of genes involved in pollen formation so that breeders can reproducibly generate male sterile plants for the production of F1 hybrids; gs of genes involved in lignin biosynthesis to facilitate paper making from vegetative tissue of the plant; gs of genes involved in flower pigment production to produce novel flower colours; gs of genes involved in regulatory pathways controlling development or environmental responses to produce plants with novel growth habit or (for example) disease resistance; elimination of toxic secondary metabolites by gs of genes required for toxin production. In addition, gs is can be useful as a means of developing virus resistant plants when the transgene is similar to a viral genome.
A major complication in the practical exploitation of this phenomenon to date is the unpredictable and low occurrence of gs. Typically there will be strong gs in as few as 5-20% of lines generated with any one construct (for examples see (28,34)). Therefore, it has not been realistic to attempt gs in plants that are difficult to transform and for which it is difficult to produce many transformants. Similarly, it would be difficult to activate gs against several different traits or against several viruses in the same plant. Even with plants that are easy to transform the need to generate multiple lines limits the ease of exploitation of gs.
The first indication that an inoculated virus could elicit gene silencing was with transgenic plants in which the transgene included cDNA of tobacco etch potyvirus (22). The lines that exhibited this virus-induced gene silencing were initially high level expressers of the transgene. After inoculation with a strain of tobacco etch potyvirus that was identical or highly similar to the transgene there was a reduction in the amount of the transgene RNA and suppression of the originally inoculated virus in the upper leaves of the plant. These upper leaves were described as having recovered because they were virus and symptom free. They were also resistant against a secondary challenge inoculation with virus that was highly similar to the transgene at the nucleotide level. All of these effects are probably due to gs at the post transcriptional level.
However, there is nothing in this report to indicate that virus-induced gene silencing is intrinsically more reproducible than any other type of gene silencing. In fact there were some lines that displayed the virus-induced gene silencing and other lines that did not (22). Furthermore there are many reports that refer to virus inoculation of transgenic plants carrying transgenes that are similar or identical to the inoculated virus. Of these reports only a minority describe virus-induced gene silencing. Thus with viral transgenes the virus induced gene silencing is the exception rather than the rule. That is, as indicated above, there was no indication from this work that gene silencing by inoculated viruses is intrinsically reproducible.
Biosource Technologies, Inc. (20,21) have suggested the use of genetic constructions based on RNA viruses which replicate in the cytoplasm of cells to provide inhibitory RNA, either anti-sense or co-suppressor RNA. Cells are transfected with the cytoplasmically-replicating genetic constructions in which the RNA encoding region is specific for the gene of interest. Experimental evidence illustrating the drawbacks and limitations of the Biosource approach is included below.
The present invention aims to overcome one or more of the many problems in the art. For instance, experimental evidence included below demonstrates with embodiments of the present invention gs is achievable more reproducibly than with conventional technology.
Briefly, the present invention in various aspects makes use of an amplicon construct which will exhibit gs targeted against sequence with homology to a sequence within the amplicon. An amplicon is a transgene DNA construct including a promoter and cDNA of at least part of a viral genome, and optionally a transcriptional terminator. Preferably, the construct includes a xe2x80x9ctargeting sequencexe2x80x9d, which may be a sequence foreign to the virus, for specifically targeting down-regulation of a gene of interest (xe2x80x9ctarget genexe2x80x9d). Further details are discussed below.
Incorporation of a construct in the genome of transgenic plants in accordance with the present invention may be used to ensure the viral cDNA is transcribed from the promoter in many or most cells of the plant, though use of a tissue- or developmentally-regulated and/or inducible promoter is possible. If the viral cis-acting elements and trans-acting factors necessary for replication are intact there will be replication of the viral RNA in the transgenic plant. As a consequence, either direct or indirect, there will be activation of gs targeted against sequences with sufficient homology to a sequence included with the replicating viral RNA, the xe2x80x9ctargeting sequencexe2x80x9d.
An amplicon construct may have an unmodified viral cDNA. In such a case the plant may be resistant against the virus as a result of gs. Other amplicon constructs may have a foreign targeting sequence inserted into the viral cDNA and the gs will be targeted against the corresponding sequence as well as against the virus. The targeting sequence may be part of a different viral genome, in which case the plant may also be resistant against this second virus. The targeting sequence may be derived from a nuclear gene or transgene, or a gene on an extrachromosomal element such as a plasmid, and the gs targeted against that gene and homologues.
The present invention will now be discussed in more detail.
According to one aspect of the present invention there is provided, preferably within a vector suitable for stable transformation of a plant cell, a DNA construct in which a promoter is operably linked to DNA for transcription in a plant cell of an RNA molecule which includes plant virus sequences which confer on the RNA molecule the ability to replicate in the cytoplasm of a plant cell following transcription. The RNA transcribed from the DNA which is under the transcriptional control of the promoter is capable of replication in the cytoplasm of a plant cell by virtue of including appropriate plant virus sequences. The transcripts, possibly including a sequence foreign to the virus, replicate as if they are viral RNAs, activating gs.
The transcribed RNA generally includes a sequence (xe2x80x9ctargeting sequencexe2x80x9d) which is complementary to a sequence in a target gene, either in the sense or anti-sense orientation, or a sequence which has sufficient homology to a target sequence for down-regulation of expression of the target gene to occur. Whilst not to be bound by any theory, it is believed that sense and anti-sense regulation involve hybridisation between sequences which are sufficiently complementary to hybridise under conditions within a cell.
The targeting sequence within the construct may be foreign to the plant virus, i.e. of or derived from a gene or sequence which the virus lacks. Those skilled in the art will understand that terms such as xe2x80x9cexogenousxe2x80x9d or xe2x80x9cheterologousxe2x80x9d may equally be used in this context.
A vector which contains the construct may be used in transformation of one or more plant cells to introduce the construct stably into the genome, so that it is stably inherited from one generation to the next. This is preferably followed by regeneration of a plant from such cells to produce a transgenic plant.
Thus, in further aspects, the present invention also provides the use of the construct or vector in production of a transgenic plant, methods of transformation of cells and plants, plant and microbial (particularly Agrobacterium) cells, and various plant products.
The function of the promoter in the amplicon construct is to ensure that the DNA is transcribed into RNA containing the viral sequences. By xe2x80x9cpromoterxe2x80x9d is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3xe2x80x2 direction on the sense strand of double-stranded DNA). A promoter xe2x80x9cdrivesxe2x80x9d transcription of an operably linked sequence.
xe2x80x9cOperably linkedxe2x80x9d means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is xe2x80x9cunder transcriptional initiation regulationxe2x80x9d of the promoter, or xe2x80x9cin functional combinationxe2x80x9d therewith.
Preferred promoters may include the 35S promoter of cauliflower mosaic virus or the nopaline synthase promoter of Agrobacterium tumefaciens (Sanders, P. R., et al (1987), Nucleic Acids Res., 15: 1543-1558). These promoters are expressed in many, if not all, cell types of many plants. Other constitutively expressed promoters may be used effectively as components of amplicon construct producing gs. Depending on the target gene of amplicon gs, other promoters including those that are developmentally regulated or inducible may be used. For example, if it is necessary to silence the target gene specifically in a particular cell type the amplicon construct may be assembled with a promoter that drives transcription only in that cell type. Similarly, if the target gene is to be silenced following a defined external stimulus the amplicon construct may incorporate a promoter that is be activated specifically by that stimulus. Promoters that are both tissue specific and inducible by specific stimuli may be used.
Inducible promoters may be advantageous in certain circumstances because they place the timing of reduction in expression of the target gene of interest under the control of the user.
The term xe2x80x9cinduciblexe2x80x9d as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is xe2x80x9cswitched onxe2x80x9d or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases upon application of the relevant stimulus by an amount effective to alter a phenotypic characteristic. Thus an inducible (or xe2x80x9cswitchablexe2x80x9d) promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero). Upon application of the stimulus, expression is increased (or switched on) to a level which brings about the desired phenotype.
Suitable promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues (Benfey et al, 1990a and 1990b) and the maize glutathione-S-transferase isoform II (GST-II-27) gene promoter which is activated in response to application of exogenous safener (WO93/01294, ICI Ltd). The GST-II-27 gene promoter has been shown to be induced by certain chemical compounds which can be applied to growing plants. The promoter is functional in both monocotyledons and dicotyledons. It can therefore be used to control gene expression in a variety of genetically modified plants, including field crops such as canola, sunflower, tobacco, sugarbeet, cotton; cereals such as wheat, barley, rice, maize, sorghum; fruit such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, and melons; and vegetables such as carrot, lettuce, cabbage and onion. The GST-II-27 promoter is also suitable for use in a variety of tissues, including roots, leaves, stems and reproductive tissues.
A construct for use in accordance with the present invention includes sequences which are DNA copies of cis-acting elements in the viral genome and of open reading frames in the viral genome that are required for replication of the viral RNA. They should be arranged in the amplicon construct so that the transcripts of the amplicon construct will replicate in the plant cells independently of other transgenes or of viruses inoculated to the plants. The cDNA copies of any other parts of the viral genome need not be included in the amplicon construct provided that their absence does not interfere with replication of the amplicon transcripts. An optional but important additional feature of some amplicon constructs in accordance with the present invention is the insertion of foreign sequences into the viral cDNA. The site of insertion of these foreign sequences is so that the foreign sequence is replicated, as RNA, as part of the viral RNA produced by transcription of the amplicon in a plant cell. The target of gene silencing is determined by the viral cDNA and any foreign targeting sequence in the amplicon construct.
The amplicon constructs used in the experimental exemplification described in this application for the purpose of illustration of aspects of the present invention without limitation are based on cDNA of the potato virus X (PVX) genome (Kavanagh, T. A., et al (1992), Virology, 189: 609-617). Many, if not all, other RNA or DNA viruses of plants may be used in generation of amplicon constructs in a manner that is similar to that described here for PVX. Particularly suitable alternatives to PVX are those viruses for which it is known that foreign sequence is tolerated as part of the replicating viral genome. Included in these examples are tobacco mosaic virus (Dawson, W. O., et al (1989), Virology, 172: 285-292), tobacco etch virus (Dolja, V. V., et al (1992), Proc. Natl. Acad. Sci. USA, 89: 10208-10212), tobacco rattle virus (Ziegler-Graff, V., et al (1991), Virology, 182: 145-155), tomato bushy stunt virus (Scholthof, H. B., et al (1993), Mol. Plant-Microbe Interact., 6: 309-322), brome mosaic virus (Mori, M., et al (1993), J. Gen. Virol., 74: 1255-1260), cauliflower mosaic virus (Futterer, J. and Hohn, T. (1991), EMBO J., 10: 3887-3896), african cassava mosaic virus (Ward, A., et al (1988), EMBO J., 7: 1583-1587), tomato golden mosaic virus. Preferred viruses for use in the present invention may be RNA viruses.
A foreign targeting sequence may be included in the construct as a substitution for a viral gene or sequence that is not required for replication or as an additional sequence added to the viral genome. A foreign sequence may be included in the amplicon as an intact open reading frame and so that it is transcribed as a subgenomic RNA. However, a foreign sequence may be included anywhere in the viral cDNA irrespective of the location of subgenomic promoter.
A sequence foreign to the viral nucleic acid and homologous or similar to a target gene of interest, may be included in a sense or anti-sense orientation in the amplicon.
In using anti-sense genes or partial gene sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a xe2x80x9creverse orientationxe2x80x9d such that transcription yields RNA which is complementary to normal mRNA transcribed from the xe2x80x9csensexe2x80x9d strand of the target gene. See, for example, Rothstein et al, 1987; Smith et al, (1988) Nature 334, 724-726; Zhang et al, (1992) The Plant Cell 4, 1575-1588, English et al., (1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Bourque, (1995), Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.
An alternative is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression. See, for example, van der Krol et al., (1990) The Plant Cell 2, 291-299; Napoli et al., (1990) The Plant Cell 2, 279-289; Zhang et al., (1992) The Plant Cell 4, 1575-1588, and U.S. Pat. No. 5,231,020.
The complete sequence corresponding to the coding sequence (in reverse orientation for anti-sense) need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding sequence to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence of a gene, e.g. a sequence that is characteristic of one or more genes in one or more pathogens against which resistance is desired, such as a regulatory sequence.
A foreign sequence may be 500 nucleotides or less, possibly about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. It may be possible to use oligonucleotides of much shorter lengths, 14-23 nucleotides, although longer fragments, and generally even longer than 500 nucleotides are preferable where possible.
It may be preferable that there is complete sequence identity in the targeting (e.g. foreign) sequence in the amplicon and the target sequence in the plant, though total complementarity or similarity of sequence is not essential. One or more nucleotides may differ in the targeting sequence from the target gene. Thus, a targeting sequence employed in a construct in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a mutant, derivative, variant or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence. A foreign sequence need not include an open reading frame or specify an RNA that would be translatable. As noted, a foreign sequence may be inserted into the amplicon construct in either orientation, for sense or anti-sense regulation. It may be preferred for there to be sufficient homology for the respective anti-sense and sense RNA molecules to hybridise. There may be gs even where there is about 5%, 10%, 15% or 20% or more mismatch between the targeting, e.g. foreign, sequence in the amplicon and the target gene.
In embodiments of the present invention which have been experimentally exemplified as described below for illustrative and non-limiting purposes only, the foreign sequence in the amplicon that determined the target of gene silencing was the uidA reporter gene (Jefferson, R. A., et al (1986), Proc. Natl. Acad. Sci. USA, 83: 8447-8451) or the gene encoding the jellyfish green fluorescent protein (Chalfie et al. (1994) Science 263: 802-805). Other disclosed example genes include tomato DWARF and arabidopsis phytoene desaturase. However, any other gene of plant, animal, fungal, bacterial or viral origin may be a target of amplicon gs provided that the corresponding foreign sequence is incorporated into the amplicon construct. Particularly suitable as targets are genes that have already been shown to be suppressible by gs in the literature. These may include, for example, chalcone synthase of petunia or polygalacturonase of tomato (Jorgensen, R. A. (1995), Science, 268: 686-691, Hamilton, A. J., et al (1995), Current Topics In i Microbiology and Immunology, 197: 77-89).
One or more targeting sequences may be included in the construct, to provide for suppression of one or more genes, e.g. when down-regulation of more than one gene is required at the same time, or for conferring resistance to more than one pathogen. A targeting sequence with sufficient homology to more than one target sequence may be used in suppressing more than one gene.
An additional optional feature of a construct used in accordance with the present invention is a transcriptional terminator. The transcriptional terminator from nopaline synthase gene of agrobacterium tumefaciens (Depicker, A., et al (1982), J. Mol. Appl. Genet., 1: 561-573) may be used, and is experimentally exemplified below. Other suitable transcriptional terminators include but are not restricted to those from soybean actin, ribulose bisphosphate carboxylase of Nicotiana plumbaginifolia (Poulson, C., et al (1986), Mol. Gen. Genet., 205: 193-200) and alpha amylase of wheat (Baulcombe, D. C., et al (1987), Mol. Gen. Genet., 209: 33-40). A transcriptional terminator sequence foreign to the virus may not be included in a construct of the invention in particular when the viral sequences included in the construct include one or more transcriptional terminator sequences.
Those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press.
Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Protocols in Molecular Biology, Second edition, Ausubel et al. eds., John Wiley and Sons, 1992.
Specific procedures and vectors previously used with wide success upon plants are described by Bevan, Nucl. Acids Res. (1984) 12, 8711-8721), and Guerineau and Mullineaux, (1993) Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148.
For introduction into a plant cell, the nucleic acid construct may be in the form of a recombinant vector, for example an Agrobacterium binary vector. Microbial, particularly bacterial and especially Agrobacterium, host cells containing a construct according to the invention or a vector which includes such a construct, particularly a binary vector suitable for stable transformation of a plant cell, are also provided by the present invention.
Nucleic acid molecules, constructs and vectors according to the present invention may be provided isolated and/or purified (i.e. from their natural environment), in substantially pure or homogeneous form, or free or substantially free of other nucleic acid. Nucleic acid according to the present invention may be wholly or partially synthetic. The term xe2x80x9cisolatexe2x80x9d encompasses all these possibilities.
An aspect of the present invention is the use of a construct or vector according to the invention in the production of a transgenic plant.
A further aspect provides a method including introducing the construct or vector into a plant cell such that the construct is stably incorporated into the genome of the cell.
Any appropriate method of plant transformation may be used to generate plant cells containing a construct within the genome in accordance with the present invention. Following transformation, plants may be regenerated from transformed plant cells and tissue.
Successfully transformed cells and/or plants, i.e. with the construct incorporated into their genome, may be selected following introduction of the nucleic acid into plant cells, optionally followed by regeneration into a plant, e.g. using one or more marker genes such as antibiotic resistance. Selectable genetic markers may be used consisting of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate.
When introducing a nucleic acid into a cell, certain considerations must be taken into account, well known to those skilled in the art. The nucleic acid to be inserted should be assembled within a construct which contains effective regulatory elements which will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material should occur. Finally, as far as plants are concerned the target cell type must be such that cells can be regenerated into whole plants.
Plants transformed with the DNA segment containing the sequence may be produced by standard techniques which are already known for the genetic manipulation of plants. DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718, NAR 12(22) 8711-87215 1984), particle or microprojectile bombardment (U.S. Pat. No. 5,100,792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al. (1987) Plant Tissue and Cell Culture, Academic Press), electroporation (EP 290395, WO 8706614 Gelvin Debeyserxe2x80x94see attached) other forms of direct DNA uptake (DE 4005152, WO 9012096, U.S. Pat. No. 4,684,611), liposome mediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol. 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U.S.A. 87: 1228 (1990d). Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech. Adv. 9: 1-11.
Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (Toriyama, et al. (1988) Bio/Technology 6, 1072-1074; Zhang, et al. (1988) Plant Cell Rep. 7, 379-384; Zhang, et al. (1988) Theor Appl Genet 76, 835-840; Shimamoto, et al. (1989) Nature 338, 274-276; Datta, et al. (1990) Bio/Technology 8, 736-740; Christou, et al. (1991) Bio/Technology 9, 957-962; Peng, et al. (1991) International Rice Research Institute, Manila, Philippines 563-574; Cao, et al. (1992) Plant Cell Rep. 11, 585-591; Li, et al. (1993) Plant Cell Rep. 12, 250-255; Rathore, et al. (1993) Plant Molecular Biology 21, 871-884; Fromm, et al. (1990) Bio/Technology 8, 833-839; Gordon-Kamm, et al. (1990) Plant Cell 2, 603-618; D""Halluin, et al. (1992) Plant Cell 4, 1495-1505; Walters, et al. (1992) Plant Molecular Biology 18, 189-200; Koziel, et al. (1993) Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology 25, 925-937; Weeks, et al. (1993) Plant Physiology 102, 1077-1084; Somers, et al. (1992) Bio/Technology 10, 1589-1594; WO92/14828). In particular, Agrobacterium mediated transformation is now emerging also as an highly efficient transformation method in monocots (Hiei et al. (1994) The Plant Journal 6, 271-282).
The generation of fertile transgenic plants has been achieved in the cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994) Current Opinion in Biotechnology 5, 158-162; Vasil, et al. (1992) Bio/Technology 10, 667-674; Vain et al., 1995, Biotechnology Advances 13 (4): 653-671; Vasil, 1996, Nature Biotechnology 14 page 702).
Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).
Following transformation, a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewd in Vasil et al., Cell Culture and Somatic Cel Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.
The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.
Also according to the invention there is provided a plant cell having incorporated into its genome a DNA construct as disclosed. A further aspect of the present invention provides a method of making such a plant cell involving introduction of a vector including the construct into a plant cell. Such introduction should be followed by recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome. RNA encoded by the introduced nucleic acid construct may then be transcribed in the cell and descendants thereof, including cells in plants regenerated from transformed material. A gene stably incorporated into the genome of a plant is passed from generation to generation to descendants of the plant, so such decendants should show the desired phenotype.
The present invention also provides a plant comprising a plant cell as disclosed.
A plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders"" Rights. It is noted that a plant need not be considered a xe2x80x9cplant varietyxe2x80x9d simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.
In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone or descendant of such a plant, or any part or propagule of said plant, off-spring, clone or descendant. Plant extracts and derivatives are also provided.
The present invention may particularly be applied in plants such as natural hosts of a plant virus, including any mentioned herein, though it is an advantage of embodiments of the present invention that viruses may be used for gene silencing in plants which are not their natural hosts, as has been demonstrated experimentally and is described below. Indeed the present inventors have demonstrated that PVX, used in many of the Examples below, can replicate in monocots in addition to its natural hosts.
The present invention may be used in plants such as crop plants, including cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorgum, millet, cassava, barley, pea and other root, tuber or seed crops. Important seed crops are oil seed rape, sugar beet, maize, sunflower, soybean and sorghum. Horticultural plants to which the present invention may be applied may include lettuce, endive and vegetable brassicas including cabbage, broccoli and cauliflower, and carnations and geraniums. The present invention may be applied to tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum, poplar, eucalyptus and pine.
As noted, transcription from the construct in the genome of a plant cell yields a cytoplasmically-replicating RNA able to down-regulate expression of a target gene in the cell, which target gene may be of the plant, endogenous to the plant genome or a transgene, or of a pathogen such as a virus.
Thus, a further aspect of the present invention provides a method which includes causing or allowing transcription from a construct as disclosed within the genome of a plant cell.
A further aspect of the present invention provides a method of reducing or suppressing or lowering the level of expression of a gene of interest (or xe2x80x9ctarget genexe2x80x9d) in a plant cell, the method including causing or allowing transcription from a construct as disclosed. Transcription produces RNA and is generally followed by replication of the transcribed RNA by virtue of the inclusion of the viral sequence. The construct may include the target gene sequence or a fragment thereof in a sense or anti-sense orientation, or a sequence with sufficient homology to the target gene sequence or a fragment thereof for the level of expression of the target gene to be reduced on production of the RNA.
As demonstrated in the Examples hereinafter, in certain embodiments the target gene may be remote from the site of the amplicon replication, which may only be activated in certain cells (for instance because the amplicon is activated by an inducible promoter). In such cases the gs may be achieved through the demonstrated systemic or remote effect. In the light of the present work, it appears that this effect may be analagous to that disclosed in Voinnet and Baulcombe (1997) Nature 389: page 553, (although in that case the initiator of the systemic gs signal was not a cytoplasmically replicating construct). Thus the distal effect may be particularly effective when the amplicon is activated in a photosynthetic xe2x80x98sourcexe2x80x99 tissue, while the target tissue is present (either locally or systemically) in xe2x80x98sinkxe2x80x99 tissue(s).
With respect to the level of expression of a gene of interest in a cell, the method will generally result in a decrease in the level of expression as compared with the level in the absence of the intervention, i.e. in comparison with equivalent wild-type cells, e.g. of plants of the same species. (Cells which are wild-type in respect of the level of expression of the gene of interest may of course not be wild-type in every respect.)
Where the target is a gene of a pathogen such as a virus, down-regulation of expression may provide an increase in resistance to the pathogen, particularly where the gene is required for, or is at least involved in, replication and/or infectivity (e.g. in an RNA-dependent RNA polymerase in a virus such as cowpea mosaic virus (Sijen, T., et al (1996), Plant Cell, 8: 2277-2294)).
The present invention will now be illustrated and exemplified with reference to experimental results and the accompanying figures. Further aspects and embodiments of the present invention, and modifications of those disclosed herein, will be apparent to those skilled in the art. All documents mentioned anywhere herein are incorporated by reference.